Growth control in mammalian cells is accomplished largely by the Rb protein regulating exit from the G1 phase (Weinberg, 1995, Cell 81, 323-330) and the p53 protein triggering growth arrest/apoptotic processes in response to cellular stress (Levine, 1997, Cell 88, 323-331). Cross-talk between these two regulatory pathways may be mediated through the p21 cdk inhibitor, which is a target of p53 transactivation as well as a factor that influences the functional status of Rb (Weinberg, 1995, Cell 81, 323-330.). An additional level of overlap between p53 and Rb is provided by the MDM2 protein that can physically associate with both proteins and prevent their growth suppression (Momand et al., 1992, Cell 69, 1237-1245; Xiao et al., 1995, Nature 375, 694-698). In tumorigenesis RB and p53 appear to serve collaborative roles as evidenced by the observations that many tumor types exhibit mutations in both RB and p53 (Williams et al., 1994, Nature Genet. 7, 480-484) and mice that are RB+/− and p53−/− develop a wider range of tumors at earlier ages than mice that are either Rb+/− or p53−/− (Williams et al., 1994, Nature Genet. 7, 480-484). Moreover, the ability of several viruses to transform cells in culture and cause tumors in mice is due to viral oncoproteins that bind to and inactivate both RB and p53 (Hawley-Nelson et al., 1989, EMBO 8, 3905-3910; Munger et al., 1989, Journal of Virology 63, 4417-4421; Mahon et al., 1987, Science 235, 1622-1628; Symonds et al., 1994, Cell 78, 703-711). The mechanistic basis for this dual requirement stems in part from the deactivation of a p53-dependent cell suicide program that would normally be brought about as a response to unchecked cellular proliferation resulting from Rb-deficiency (Ko and Prives, 1996, Genes Devel. 10, 1054-1072; Gottlieb and Oren, 1996, Biophysica Acta. 1287, 77-102; Levine, 1997, Cell 88, 323-331).
p53 mutation is thought to be the most frequent genetic alteration in human cancers (Hollstein et al., 1991, Science 253, 49-53; Levine et al., 1991, Nature 351, 453-456). In proliferating normal and neoplastic cells, the consequences of p53 over-expression are context-dependent, resulting in either cell cycle arrest or induction of apoptosis (Ko and Prives, 1996, Genes Devel. 10, 1054-1072). These biological endpoints provide a basis for p53's anti-oncogenic actions (Eliyahu et al., 1989, Proc. Natl. Acad. Sci. USA 86, 8763-8767; Finlay et al., 1989, Cell 57, 1083-1093) and have been shown to relate to its capacity to function as a sequence-specific transcription factor (Pietenpol et al., 1994, Proc. Natl. Acad. Sci. USA 91, 1998-2002; Crook et al., 1994, Cell 79, 817-827), and to interact with key cellular proteins. The critical role served by p53 in these diverse physiological processes necessitates that p53 activity be subject to stringent multi-level regulation. One crucial level of regulation involves the MDM2 protein whose direct interaction with p53 blocks p53-mediated transactivation (Chen et al., 1995, Mol. Med 1, 141-142) and targets the p53 protein for rapid degradation (Levine, 1997, Cell 88, 323-331; Kubbutat et al., 1997, Nature 387, 299-303; Haupt et al., 1997, Nature 387, 296-299). MDM2 itself has been shown to be amplified in primary tumors (Oliner et al., 1992, Nature 362, 857-860), to act as an immortalizing oncogene in cell culture (Finlay, 1993, Molecular & Cellular Biology 13, 301-306), and to directly repress basal transcription (Thut et al., 1997, Genes Devel. 11, 1974-1986).
In human cancers, disruption of the RB pathway can result from inactivation of RB itself through gene mutation/deletion, viral sequestration or hyperphosphorylation (Weinberg, 1995, Cell 81, 323-330), or through disregulation of the components controlling the degree of RB phosphorylation. The latter can take place through activating mutations in the G1 specific Cyclin-Dependent Kinase 4 (CDK4) catalytic unit, up-regulation of D-type cyclin levels, and/or elimination of INK4s (for INhibitors of Cyclin-Dependent Kinase 4) (Sherr, 1996, Science 274, 1672-1676). The products of INK4 family genes have been shown to bind to CDK4 and inhibit CDK4-directed phosphorylation of Rb (Quelle, et al., 1995a, Oncogene 11, 635-645; Serrano et al., 1993, Nature 366, 704-707), thereby blocking exit from the G1 phase of the cell cycle (Sherr, 1996, Science 274, 1672-1676). One member of the INK4 family, INK4a, has been shown to exhibit loss of function in a wide spectrum of tumor types; this pathogenetic event appears to be exceeded in frequency only by p53 inactivation. The basis for the prominence of INK4a, as opposed to other members of the INK4 family, in tumor suppression is not fully understood but may relate to its unusual capacity to encode two distinct proteins—the cyclin-dependent kinase inhibitor, p16INK4a, and a novel protein of unknown function, p19ARF. This special feature of INK4a results from a unique gene organization in which the two INK4a gene products are encoded by different first exons and alternative reading frames residing in a common second exon. The fact that both gene products are often eliminated or mutated in many cancers has raised questions regarding their relative contributions to INK4a-mediated tumor suppression.
Compelling support for p16INK4a as a critical target of tumorigenesis includes germline mutations/deletions exclusively affecting the p16INK4a ORF in melanoma-prone kindreds and a tumor-associated CDK4 mutation rendering this kinase insensitive to p16INK4a inhibition (Zuo et al., 1996, Nature Genet. 12, 97-99). With regard to p19ARF, although direct evidence linking loss of p19ARF function with human tumorigenesis has been lacking, many INK4a mutations/deletions map to the exon 2 region that is shared by p19ARF and a p19ARF-specific knockout leads to spontaneous tumor formation in mice (Kamijo et al., 1997a, Cell 91, 649-659).
Some clues addressing p19ARF's mechanism of action have been provided by the requirement for p53 in p19ARF-induced G1 arrest and by an absence of p53 mutations in post-crisis p19ARF−/− MEF cultures (Kamijo et al., 1997a, Cell 91, 649-659) and in RAS-induced melanomas arising in the INK4a null mice (Chin et al., 1997, Genes and Development 11, 2822-2834). Additionally, studies reported here suggest that p19ARF requires p53 function to suppress cellular transformation. All of these observations have led to the intriguing possibility that the INK4a gene is linked not only to the Rb pathway through p16INK4a but also to the p53 pathway through p19ARF.
The present invention describes the determination of the function of the novel protein p19ARF. The inventors have determined that the novel p19ARF protein acts as a suppressor of oncogenic transformation. The inventors specifically ascertained that p19ARF engages the p53 pathway through physical interactions with the MDM2 oncoprotein. p19ARF specifically inhibits the oncogenic actions of MDM2, blocks MDM2-induced degradation of p53, and enhances p53-dependent transactivation. The inventors additionally demonstrated that loss of INK4a attenuates apoptosis brought about by Rb deficiency. These studies provide physical and mechanistic insight fortifying INK4a's position at the nexus of the two most important tumor suppressor pathways governing the development of neoplasia, and provide an explanation for the frequent involvement of INK4a in tumorigenesis.